Differential effects of progestogens on HIV-1 replication and host gene expression in primary PBMCs and cervical tissue explants

Includes bibliographical referencesThe synthetic progestogens, medroxyprogesterone acetate (MPA) and norethisterone enanthate (NET-EN),are widely used in developing countries as injectable contraceptives, where disease burden is high.Levonorgestrel (LNG) is a common progestogen used in oral contraceptives and intrauterine devices. Some studies suggest that MPA, unlike NET, increases HIV-1 acquisition in women, while few studies have reported on the effects of LNG on HIV-1 acquisition. Whether MPA, NET and LNG differentially affect HIV-1infection and the expression of key genes relevant to HIV-1 acquisition via differential molecular mechanisms is key to understanding choice of progestogen contraceptive in young women at high risk forHIV-1 acquisition. The central hypothesis of this study is that the differential effects on host gene expression and HIV-1replication by the different progestogens is due to their differential selectivity to towards different steroid receptors, in particular the glucocorticoid receptor (GR). In order to investigate this hypothesis, the regulation of selected genes was investigated in cervical tissue explants from premenopausal, HIV-1negative, and contraception negative women and peripheral blood mononuclear cells (PBMCs) from women, by real time quantitative PCR, western blotting and Luminex assays, in response to physiologically relevant doses of progestogens. Infection assays were performed in the absence and presence of HIV-1using HIV-1BAL-RENILLA or HIVpNL4.3 infectious molecular clones (IMCs). The GR antagonist RU486 or GR siRNA knockdown was used to determine the role of the GR in modulating ligand-specific effects. PBMCs and primary cervical explants were chosen as useful models to assess the direct effects of these progestogens in both the systemic and in the local mucosal immune environments. In PBMCs, MPA like dexamethasone (DEX, a GR specific agonist), showed anti-inflammatory effects, decreasing pro-inflammatory interleukin (IL) 6, IL8 and regulated upon activation, normal T cell expressed and secreted(RANTES) levels and increasing anti-inflammatory glucocorticoid interacting leucine zipper (GILZ) gene expression levels, while NET, progesterone (P4) and LNG did not, after 48 hours. In primary ectocervical tissue explants, DEX, cortisol, MPA and P4 significantly repressed IL6 while only DEX, cortisol and MPA significantly repressed IL8 and increased GILZ gene expression levels after 48 hours. Steroid receptor expression was characterised in both PBMCs and ectocervical explants. GR was the only detectable steroid receptor protein expressed in PBMCs, while ectocervical explants expressed all the steroid receptors. The progesterone and estrogen receptor levels were higher in ectocervical explants from donors that were in the follicular phase compared to ectocervical explants from donors in the luteal phase of the menstrual cycle. In PBMCs, results suggested that differential gene expression by MPA versus NET and P4is mediated via the GR after 48 hours. Furthermore, it was observed that MPA and DEX, unlike NET, LNG and P4 increases HIV-1 replication in viable PBMCs, in the majority of donors. The increase in HIV-1replication in the MPA treated PBMC samples correlated significantly with an increased in IL6 mRNA levels